NEMATODE RESISTANCE IN TRANSGENIC RICE PLANTS By Elif Ezgi Inan

What is a nematode? Nematodes are a diverse animal phylum that can live in a broad range of environments. They are the most numerous multicellular animals on earth.

As I looked up online I found that rice is one of the most affected crops. Nematodes effect on rice cause a huge amount of damage on the economy.

Meloidogyne graminicola (rice root knot nematode) M. graminicola is considered to be a major threat to rice agriculture. It is a devastating plant pathogen, and is therefore classified as a quarantine pest in many countries. Rice is the most important host for this nematode, but it has a wide range of alternative hosts.

How nematodes manipulate plant development pathways for infection: Nematodes secrete effector proteins to reprogramme root cells and counteract defense. Nematode effector proteins modulate auxin flow by targeting auxin transport proteins. Nematode infection affects the localization and activity of PIN proteins. Nematodes secrete plant peptide mimics to affect cell differentiation. PIN PROTEIN AUXIN

Symptoms of this root-knot nematode M. graminicola can be detected when plants are uprooted as it causes swellings and galls throughout the root system. Infected root tips become swollen and hooked, a symptom which is especially characteristic of this nematode.

Left: Female nematodes and eggs inside rice root gall. Right: Characteristic hooked, root tip galls on rice.

DESIGN A plasmid model that gains nematode resistance by the help of cysteine protease inhibitors. Cysteine proteases work as an anthelmintic (antiparasitic drug) towards the root-knot nematode. The reason why they affect Meloidogyne graminicola is because they are the only class of proteinases not expressed in the digestive system of mammals. The specific inhibitor of cysteine proteinases, blocks this activity completely, indicating that it was essentially mediated by cysteine proteinases. Beta-glucuronidase, is used as the reporter gene to monitor gene expression and finally the hygromycin (a type of antibiotic) resistance gene to make the marker resistance to antibiotics.

To transfer these genes, gene gun is used instead of transferring with bacteria . The reason for that is because, according to previous researches about nematode resistance in rice, transferring to rice using bacteria doesn’t give any significant result.

How the Gene Gun Works: The gene gun, also known as the particle bombardment device, was developed to enable penetration of the cell wall so that genetic material containing a gene of interest can be transferred into the cell. This technique is most suitable for those plants which hardly regenerate and do not show sufficient response to gene transfer through Agrobacterium for example, rice, wheat corn and chickpea

The particle bombardment method starts with coating gold particles with plasmid DNA. The coated particles are coated on a macro- projectile, which is accelerated with air pressure and shot into the nucleus of the plant tissue taken from plants on a petri plate. These undifferentiated cells are grown before performing the transformation. When the gold particle land into the nucleus the genes dissolve away from it and then incorporate onto the DNA of the chromosomes. DNA allows the new genes without replacing any genes.

There is the vacuum chamber where all the stuff goes There is the vacuum chamber where all the stuff goes. Up there, there is the pressure chamber where the helium build up pressure to a certain point and creating a shockwave that will project the DNA down into the cell. Before doing that process to test that we have the genes, onion can be used. Onion is placed in the plate and after the bombardment of the cell we can see whether the cells are attached to the onion cells or not. The reason why onion is used is because its cells are decent size so they are easy to detect.

Ten-to fifteen-day-old immature and sterilized seeds are  removed and plated on the appropriate medium for 2 days before bombardment. The plasmid that has the cysteine protease inhibitor gene (which is used as the defence mechanism), Beta-glucuronidase (which is used as the reporter gene) and hygromycin (an antibiotic) resistance gene to make the marker resistant to antibiotics.

Particle bombardment is carried out. Two days after bombardment, immature embryos are subcultured onto the appropriate medium supplemented with hygromycin. After some time, plants are regenerated. Polymerase Chain Reaction is carried out. Beta-glucuronidase activity is used to see whether the promoter worked or not. The western blot technique is used to test if the specific proteins we need are in the plasmids. Samples of regenerated plants are placed in a nematode present environment

Presence of cysteine proteinase inhibitor gene presence of Beta-glucuronidase gene presence of hygromycin gene Presence of hygromycin in the plate where tissues are kept after regeneration Meloidogyne graminicola resistance 1

Other Control Techniques: Most of the control measures of nematodes are too heavily dependant on environmentally hazardous nematicides. These nematicides need to be used on the plant in a certain amount of time and they are not effective on all of the species of nematodes. The cost of these chemicals is excessive and using them for once is not enough, they need to be used over and over again.

Here are some chemical-free prevention methods but again they are not efficient in long-term and they cannot be applied to all types of crops: Flooding: Damage to the crop can be avoided by raising rice seedlings in flooded soils thus preventing root invasion by the nematodes. Crop Rotation: Certain crops are resistant or poor hosts of M. graminicola and could be used in rotation to reduce nematode populations. Long rotations, greater than 12 months, will be needed to reduce M. graminicola soil populations to low levels Increasing soil fertility can compensate for some damage.

Advantages: It is a green solution for controlling nematodes. No harmful chemicals are used. It is applied only once so the it doesn’t need to be controlled all the time. Disadvantages: The whole process is expensive especially the gene gun. It may not give positive result every time applied. Experienced scientists are needed. Nematode infections can be encountered anywhere in the world so it may not be accessible for everyone. It is not seen as a priority right now.

Sources http://www.sciencedirect.com/science/article/pii/S1369526611000240?showall%3D true%26via%3Dihub https://en.wikipedia.org/wiki/PIN_proteins https://en.wikipedia.org/wiki/Integral_membrane_protein https://en.wikipedia.org/wiki/Polar_auxin_transport https://www.slideshare.net/faisaltnau/nematode-resistance-faisal http://www.cabi.org/isc/datasheet/33243 http://nematode.unl.edu/what-is-a-nematode.htm https://www.researchgate.net/publication/225824092_Expression_of_an_engineered _cysteine_proteinase_inhibitor_Oryzacystatin- IDD86_for_nematode_resistance_in_transgenic_rice_plants https://www.google.com/search?q=auxin&rlz=1C5CHFA_enTR732TR732&source=lnm s&tbm=isch&sa=X&ved=0ahUKEwjH5- Cl_LvVAhUM2IMKHRCmD4AQ_AUICigB&biw=1182&bih=596#imgrc=z1SK6nSJys36-M: https://en.wikipedia.org/wiki/Agrobacterium#Uses_in_biotechnology

THANK YOU